Limits...
Sublethal Concentrations of Antibiotics Cause Shift to Anaerobic Metabolism in Listeria monocytogenes and Induce Phenotypes Linked to Antibiotic Tolerance.

Knudsen GM, Fromberg A, Ng Y, Gram L - Front Microbiol (2016)

Bottom Line: A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance.However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested.Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Technical University of Denmark Kongens Lyngby, Denmark.

ABSTRACT
The human pathogenic bacterium Listeria monocytogenes is exposed to antibiotics both during clinical treatment and in its saprophytic lifestyle. As one of the keys to successful treatment is continued antibiotic sensitivity, the purpose of this study was to determine if exposure to sublethal antibiotic concentrations would affect the bacterial physiology and induce antibiotic tolerance. Transcriptomic analyses demonstrated that each of the four antibiotics tested caused an antibiotic-specific gene expression pattern related to mode-of-action of the particular antibiotic. All four antibiotics caused the same changes in expression of several metabolic genes indicating a shift from aerobic to anaerobic metabolism and higher ethanol production. A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance. However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested. This shift in metabolism could be a survival strategy in response to antibiotics to avoid generation of ROS production from respiration by oxidation of NADH through ethanol production. The monocin locus encoding a cryptic prophage was induced by co-trimoxazole and repressed by ampicillin and gentamicin, and this correlated with an observed antibiotic-dependent biofilm formation. A monocin mutant (ΔlmaDCBA) had increased biofilm formation when exposed to increasing concentration of co-trimoxazole similar to the wild type, but was more tolerant to killing by co-trimoxazole and ampicillin. Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

No MeSH data available.


Related in: MedlinePlus

Killing of L. monocytogenes wild type EGD (■, black full line), Δlmo1634 (▲, black broken line), Δlmo1179 (●, gray full line) and Δlmo1634/Δlmo1179 (●, gray broken line) mutants with ampicillin (A), tetracycline (B), co-trimoxazole (C), or gentamicin (D) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 3 μg/ml ampicillin (A), 3.5 μg/ml tetracycline (B), 10 μg/ml co-trimoxazole (C), or 30 μg/ml gentamicin (D). The experiment was performed with three biological replicates and error bar are standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940397&req=5

Figure 3: Killing of L. monocytogenes wild type EGD (■, black full line), Δlmo1634 (▲, black broken line), Δlmo1179 (●, gray full line) and Δlmo1634/Δlmo1179 (●, gray broken line) mutants with ampicillin (A), tetracycline (B), co-trimoxazole (C), or gentamicin (D) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 3 μg/ml ampicillin (A), 3.5 μg/ml tetracycline (B), 10 μg/ml co-trimoxazole (C), or 30 μg/ml gentamicin (D). The experiment was performed with three biological replicates and error bar are standard deviation.

Mentions: The induction levels of lmo1634 during antibiotic exposure, led us to hypothesize that lmo1634 is playing a role in antibiotic tolerance by causing increased production of ethanol which is normally only produced under anaerobic conditions (Romick et al., 1996). We expected the Δlmo1634 mutant to have altered antibiotic susceptibility when compared to the wild type EGD, however, this was not the case (p = 0.18–0.72 at 72 h; Figures 3A–D). We speculated this could be due to redundancy between lmo1634 and other genes with the same function. Three other genes are annotated as ADH genes in the genome of L. monocytogenes EGD-e of which two are of different protein families (lmo0773 and lmo2836). A blastp show high homology (E-value 2e-129 and 44% identity over 52% coverage) between the third annotated ADH (lmo1179 or eutE) and the ALDH domain of lmo1634 and more likely encode an aldehyde oxidoreductase. The ADH domain of lmo1634 has three homologies (eutG or lmo1171, lmo1166, and lmo1165) with E-values between 4e-44 and 2e-88 (between 31–41% identity with 45–46% coverage). These four genes (lmo1165, lmo1166, lmo1171, and lmo1179) are all located in the locus encoding the vitamin B12 biosynthesis genes and utilization of propanediol and ethanolamine. With several possible genes being redundant of the ADHs, we deleted the lmo1179 to force the mutant to reroute to ethanol production by either of the other ADHs. Both the Δlmo1179 mutant and the Δlmo1634/Δlmo1179 double mutant were indeed more tolerant to killing with high concentration of ampicillin (p = 0.004 and 0.08 at 72 h, respectively; Figure 3A), tetracycline (p = 0.043 and 0.047 at 72 h; Figure 3B) and co-trimoxazole (both p = 0.0002 at 72 h; Figure 3C) but not to gentamicin (p = 0.99 and 0.23 at 72 h, respectively; Figure 3D). A similar pattern were observed when grown in a MIC growth assay where MIC was twofold higher for the Δlmo1179 mutant and the Δlmo1634/Δlmo1179 double mutant (data not shown) when grown with tetracycline and co-trimoxazole. Both the single and double mutant grew more poorly than the wild type with a generation times of 131 and 149 min, respectively, compared to the wildtype at 103 min when grown in BHI broth without antibiotics at 37°C (Supplementary Figure S4).


Sublethal Concentrations of Antibiotics Cause Shift to Anaerobic Metabolism in Listeria monocytogenes and Induce Phenotypes Linked to Antibiotic Tolerance.

Knudsen GM, Fromberg A, Ng Y, Gram L - Front Microbiol (2016)

Killing of L. monocytogenes wild type EGD (■, black full line), Δlmo1634 (▲, black broken line), Δlmo1179 (●, gray full line) and Δlmo1634/Δlmo1179 (●, gray broken line) mutants with ampicillin (A), tetracycline (B), co-trimoxazole (C), or gentamicin (D) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 3 μg/ml ampicillin (A), 3.5 μg/ml tetracycline (B), 10 μg/ml co-trimoxazole (C), or 30 μg/ml gentamicin (D). The experiment was performed with three biological replicates and error bar are standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940397&req=5

Figure 3: Killing of L. monocytogenes wild type EGD (■, black full line), Δlmo1634 (▲, black broken line), Δlmo1179 (●, gray full line) and Δlmo1634/Δlmo1179 (●, gray broken line) mutants with ampicillin (A), tetracycline (B), co-trimoxazole (C), or gentamicin (D) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 3 μg/ml ampicillin (A), 3.5 μg/ml tetracycline (B), 10 μg/ml co-trimoxazole (C), or 30 μg/ml gentamicin (D). The experiment was performed with three biological replicates and error bar are standard deviation.
Mentions: The induction levels of lmo1634 during antibiotic exposure, led us to hypothesize that lmo1634 is playing a role in antibiotic tolerance by causing increased production of ethanol which is normally only produced under anaerobic conditions (Romick et al., 1996). We expected the Δlmo1634 mutant to have altered antibiotic susceptibility when compared to the wild type EGD, however, this was not the case (p = 0.18–0.72 at 72 h; Figures 3A–D). We speculated this could be due to redundancy between lmo1634 and other genes with the same function. Three other genes are annotated as ADH genes in the genome of L. monocytogenes EGD-e of which two are of different protein families (lmo0773 and lmo2836). A blastp show high homology (E-value 2e-129 and 44% identity over 52% coverage) between the third annotated ADH (lmo1179 or eutE) and the ALDH domain of lmo1634 and more likely encode an aldehyde oxidoreductase. The ADH domain of lmo1634 has three homologies (eutG or lmo1171, lmo1166, and lmo1165) with E-values between 4e-44 and 2e-88 (between 31–41% identity with 45–46% coverage). These four genes (lmo1165, lmo1166, lmo1171, and lmo1179) are all located in the locus encoding the vitamin B12 biosynthesis genes and utilization of propanediol and ethanolamine. With several possible genes being redundant of the ADHs, we deleted the lmo1179 to force the mutant to reroute to ethanol production by either of the other ADHs. Both the Δlmo1179 mutant and the Δlmo1634/Δlmo1179 double mutant were indeed more tolerant to killing with high concentration of ampicillin (p = 0.004 and 0.08 at 72 h, respectively; Figure 3A), tetracycline (p = 0.043 and 0.047 at 72 h; Figure 3B) and co-trimoxazole (both p = 0.0002 at 72 h; Figure 3C) but not to gentamicin (p = 0.99 and 0.23 at 72 h, respectively; Figure 3D). A similar pattern were observed when grown in a MIC growth assay where MIC was twofold higher for the Δlmo1179 mutant and the Δlmo1634/Δlmo1179 double mutant (data not shown) when grown with tetracycline and co-trimoxazole. Both the single and double mutant grew more poorly than the wild type with a generation times of 131 and 149 min, respectively, compared to the wildtype at 103 min when grown in BHI broth without antibiotics at 37°C (Supplementary Figure S4).

Bottom Line: A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance.However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested.Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Technical University of Denmark Kongens Lyngby, Denmark.

ABSTRACT
The human pathogenic bacterium Listeria monocytogenes is exposed to antibiotics both during clinical treatment and in its saprophytic lifestyle. As one of the keys to successful treatment is continued antibiotic sensitivity, the purpose of this study was to determine if exposure to sublethal antibiotic concentrations would affect the bacterial physiology and induce antibiotic tolerance. Transcriptomic analyses demonstrated that each of the four antibiotics tested caused an antibiotic-specific gene expression pattern related to mode-of-action of the particular antibiotic. All four antibiotics caused the same changes in expression of several metabolic genes indicating a shift from aerobic to anaerobic metabolism and higher ethanol production. A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance. However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested. This shift in metabolism could be a survival strategy in response to antibiotics to avoid generation of ROS production from respiration by oxidation of NADH through ethanol production. The monocin locus encoding a cryptic prophage was induced by co-trimoxazole and repressed by ampicillin and gentamicin, and this correlated with an observed antibiotic-dependent biofilm formation. A monocin mutant (ΔlmaDCBA) had increased biofilm formation when exposed to increasing concentration of co-trimoxazole similar to the wild type, but was more tolerant to killing by co-trimoxazole and ampicillin. Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

No MeSH data available.


Related in: MedlinePlus